This R21 proposal is to determine if Krppel-like factors (KLFs) promote functional maturation of stem cell- derived cardiomyocytes, and to establish the physiological and molecular genetic basis for the maturational effects. An important impediment to realizing the promise of hESC and hiPSC-derived cardiomyocytes for drug discovery, research and myocardial regeneration is that stem cell-derived cardiomyocytes exhibit electrical and mechanical properties of fetal, rather than adult, cells. Critically, immatue cardiomyocytes do not generate the force of mature myocytes, and their electrical properties are potentially arrhythmogenic in a transplant setting. Moreover, immature ion channel and physiological properties diminish the predictive value of in vitro analyses such as for assessing adverse cardiac effects of drug candidates. Although efficient protocols are now used to derive cardiomyocytes from stem cells, even the best yield immature, fetal-like cells since very little is known about the signals that direct maturation. Data are presented showing the development of instrumentation and software for high throughput assessment of electrophysiological maturation. Using this technology, we provide preliminary evidence that KLFs can promote electrical and ion channel profile maturation of hESC-derived and neonatal rat ventricular cardiomyocytes. The specific aims of this proposal are to: 1) define the KLF-induced maturation by functional expression of ion channels and pumps, drug sensitivity profiling, action potential and calcium handling metrics, contractile apparatus structure, and force generation; and 2) establish the molecular genetic basis for the physiological effects, in part by determining the binding partners and gene targets of KLFs by mass spectroscopic and next generation ChIPSeq analyses. The identification of signals that promote cardiomyocyte maturation should lead to improved methods for deriving functional cardiomyocytes that will be enabling for many stem cell applications in basic and applied research. Moreover, the ability to direct exogenous or endogenous stem cell sources will be essential to achieve functional regeneration of the heart.